Measurement of photosynthetic activity that occurs in photosynthetic organisms such as phytoplankton or higher plants is important to understanding phytoplankton and higher plants basic physiology, as well as in ecological studies of the environmental stress. For instance, in ocean studies, measurement of photosynthesis of phytoplankton is useful in understanding the ocean carbon cycle and predicting how climate-induced changes in ocean circulation, as well as, anthropogenic perturbation affect ocean productivity, and vice versa, how the oceans can mediate the climate change. Assessment of photosynthesis by photosynthetic organisms requires either a direct measurement, or an indirect approach based on measurement of photosynthetic parameters.
Direct measurements of photosynthesis of phytoplankton or higher plants include those of CO.sub.2 exchange, O.sub.2 evolution, or radioactive labelled carbon incorporation (i.e., .sup.14 C method). However, these measurements are laborious, time consuming, and not applicable in certain conditions. In studying phytoplankton, the .sup.14 C measurement method requires an incubation and can be done only for discrete, bottled samples. Further, accuracy in photosynthesis measurements of phytoplankton in laboratory settings are limited as a result of removal of the phytoplankton from its normal ambient nutrient flux, and laboratory simulation of ambient light and temperature conditions.
Indirect measurements of photosynthesis, based on a functional relationship between photosynthetic activity and fluorescence, have proven to be more successful. Such indirect measurement methods include both passive fluorescence and active fluorescence techniques. Passive fluorescence techniques are based on measurement of solar induced fluorescence and utilize photosensors for detecting both fluorescence and ambient solar irradiance.
An example employing the passive fluorescence technique is described in U.S. Pat. No. 4,804,849 granted to Booth et al. which discloses an apparatus for optically measuring scalar irradiance or incident flux of radiant energy and for optically measuring naturally occurring chlorophyll fluorescence or upwelling radiance from photosynthetic organisms in a parcel of water in a natural setting. Computer means are used for comparing those two measurements and for determining the concentrations of chlorophyll and calculating the rate of primary photosynthetic production. Unfortunately, passive fluorescence techniques are flawed by an assumption that the ratio of the photosynthetic to fluorescence yield is constant. In nature, this ratio can vary by as much as 10:1, making the passive based estimates of photosynthesis unreliable. More detailed measurement and study of photosynthetic processes, such as light absorption, primary photochemistry, and electron transport between so-called Photosystem II (PSII), and Photosystem I (PSI), are not possible with passive fluorescence techniques.
Active fluorescence techniques, on the other hand, are based on flash stimulated fluorescence. An example employing an active fluorescence technique, is contained in U.S. Pat. No. 4,650,336 granted to Moll which discloses a method and device for measuring photosynthesis, specifically variable fluorescence of plants. Variable fluorescence is measured as the difference between a low level, steady state fluorescence and a higher level of a fluorescent transient. The fluorometer device disclosed by Moll has one lamp to provide constant-level light to bring about continuous, steady state fluorescence of a plant, and a flash lamp to provide a flash of light (excitation energy) to bring about a transient fluorescence of the plant. The device and method of Moll utilize the second flash lamp to produces either a single flash, or series of flashes at slow repetition rate, approximately one hundred (100) Hz. Even at 100 Hz the flash rate is too slow to effectively measure the faster photosynthetic processes occurring in photosynthetic organisms.
Another active fluorescence technique is described in our earlier U.S. Pat. No. 4,942,303. That technique enables more detailed measurement of photosynthesis. Specifically, our active fluorescence technique involves use of "pump and probe" flashes for measuring the change in fluorescence of phytoplankton or higher plants. A relatively low intensity probe flash is followed quickly by a pump flash that is usually made intense enough to saturate PSII. Also disclosed in that earlier patent is a computer controlled fluorometer device and method that efficiently and accurately measures photosynthesis by precisely monitoring and recording changes in fluorescence produced by a computer controlled series of cycles of probe and pump flashes. From these measurements various photosynthetic parameters relating to the faster photosynthetic processes can be determined and incorporated into a mechanistic model of photochemistry based on the kinetics of electron flow between Photosystems II and I. The pump and probe technique, although very successful in measuring the photosynthesis occurring in phytoplankton or higher plants, has the following operational limitations:
1. In order to measure the absorption cross-section and the rate of electron flow from PSII to PSI the pump and probe fluorometer employs a sequence of probe, pump, and probe flashes, repeated up to 30 times, with the intensity of the pump flash changed from zero to a supersaturating level, or with the delay between the pump, and the second probe flash changing from 80 .mu.s to 300 ms. These two protocols require 5 minutes to 10 minutes of fluorometer operation in order to make appropriate measurements. Particularly, when the pump and probe technique is used in a profiling mode for studying phytoplankton in the ocean, where these protocols often have to be executed at every meter of a water column, the time required for making the measurements is prohibitively long. PA1 2. The intensity of the probe flash has to be kept below 1% of the PSII saturation level. This low intensity flash results in a low signal to noise ratio, particularly at low chlorophyll concentrations. PA1 3. The pump and probe fluorometer requires two separate excitation channels (i.e., two flashers) which complicates construction, and increases the cost of the fluorometer. PA1 4. Execution of a full experimental protocol, particularly in studying phytoplankton in the ocean, utilizes a large amount of electrical power. This requirements limits long-term, remote mooring applications where electrical batteries are used to power the fluorometer.
Another factor limiting active fluorescence techniques for study and measurement of photosynthesis is the current state of the art of flash lamps and flasher circuits. Specifically, many state of the art flash lamps and flasher circuits operate at too slow a repetition rate to permit accurate and rapid measurement of the faster (100 .mu.s to 300 ms) photosynthetic processes.
For studying photosynthesis, xenon flash lamps are a preferred source of excitation light for use in active fluorescence techniques because of their ability to produce a bright and very broad spectral range of light with high efficiency. Xenon flash lamps typically are operated to generate a flash of light when energy stored in an associated capacitor is discharged. Discharge is initiated by a triggering spark generated by a high voltage (5-15 kV) pulse prior to the flash. Generally, once triggered the flash lamp uses all the energy stored in the discharge capacitor. The light intensity of the flash is proportional to the stored energy, E, which is a function of capacitance, C, and the voltage, V, (E=0.5 V.sup.2 C). The energy of the flash can be controlled by varying the voltage on the discharge capacitor. To generate a subsequent flash, it is necessary to recharge the capacitor, which requires prior cooling of the plasma in the flash lamp. This limits the frequency of flashes to less than one hundred (100) Hz, or 10 ms minimum time delay between pulses (Cramer and Crofts. Photosynthesis Research 23, 231-240, 1990).
One attempt to produce a series of flashes at a rate faster than one hundred (100) Hz has included a bank, or a plurality, of flash lamps each with separate discharge capacitors. This solution requires complicated optics for directing the flash light onto a target or sample. Although a high rate of flashes can be produced by suitably sequencing ignition of the bank of flash lamps, there is an upper limit achievable based on the number of flash lamps. Additionally, the energy of the flashes cannot be changed unless a separate high voltage power supply with controlled voltages is used. This approach results in a large, complicated and costly fluorometer design.
In the field of cameras, flashing circuits have been devised using linear xenon flash lamps and Insulated Gate Biploar Transistors (IGBT) to more efficiently control a single high speed flash that is useful for photographing a subject. In these applications, control of the time for switching the flash lamp ON and OFF permits illumination of a photographed object with a precisely dosed amount of light. Usually, after switching the lamp ON, a photodiode measures the amount of light delivered to the photographed object, and generates an OFF signal after sufficient exposure. In these applications the triggering signal is either generated by the same type of IGBT transistor, as disclosed in U.S. Pat. No. 5,187,410 granted to Shimizu et al., or by using a standard thyristor circuit as disclosed in U.S. Pat. No. 5,159,381 granted to Harrison. Other examples, such as U.S. Pat. No. 5,107,292 granted to Tanaka et al. and U.S. Pat. No. 5,184,171 granted to Uenishi, describe the use of an IGBT that is switched ON before the triggering signal, thus reducing the high voltage on the collector of the IGBT before switching ON. In U.S. Pat. No. 5,075,714 granted to Hagiuda et al., an IGBT switch allows a doubling of the high voltage of the flash lamp at the end of the lamp ON state, thus reducing the high voltage rating of the charging circuitry. Another example U.S. Pat. No. 5,130,738 granted to Hirata uses an IGBT to deionize a flash lamp following switching of the lamp OFF. Switching the lamp OFF with an IGBT after a flash, stops the current flow through the lamp, allowing both the deionization of the lamp and instant recharging of the discharge capacitor. These examples disclose circuitry for delivering a single flash, and none of the examples disclose circuitry for producing and controlling a series of fast repetition rate flashes.
In another invention in U.S. Pat. No. 5,180,953 granted to Hirata et al., a strobe device uses a step-up capacitor connected to a flash discharge tube which is charged by current flowing through the tube. Thus, the device realizes a rapid charging of its step-up capacitor resulting in repeated high-speed luminous emissions of several tens of Hz (i.e., approximately 20 Hz to 30 Hz), which is not nearly fast enough for studying the faster processes of photosynthesis.
Thus, there is the need for a Fast Repetition Rate Fluorometer operable to produce a series of fast repetition rate flashes in the range of the faster processes of photosynthesis, i.e. 10,000 Hz to 250,000 Hz and at controlled energies sufficient to gradually and incrementally effect the faster photosynthetic processes occurring in PSII and PSI in phytoplankton or higher plants, for accurate and rapid measurement of fluorescence with high signal to noise ratios and determination of photosynthetic parameters.